The T-cell receptor (TCR) alpha/delta locus represents a fascinating and valuable model to evaluate the mechanisms by which highly localized and developmental stage-specific control is imparted to the processes of V(D)J recombination and transcription. It is proposed that these processes are orchestrated not only by positively acting cis-regulatory elements such as the TCR delta (E delta) and TCR alpha (E alpha) enhances, but in addition , by enhancer-blocking elements that restrict the influence of these enhancers to defined regions of the locus. Versions of a previously described transgenic TCR delta minilocus V(D)J recombination reporter, as well as wild-type and genetically manipulated versions of the endogenous TCR alpha/delta locus, will be used to explore the molecular basis for developmental control. Gene targeting will be used to determine whether a novel enhancer-blocking element, BEAD-1, functions in vivo to prevent E delta from activating J alpha chromatin in DN thymocytes. The molecular basis for enhance-blocking will be investigated by the identification of functionally important cis-elements of BEAD-1, and the proteins that bind to these sites. The molecular basis for developmental activation of E alpha at the DN to DP transition will be addressed by analysis of developmental changes in E alpha occupancy and function in versions of the minilocus and the endogenous locus. Models for developmental inactivation of E delta will be similarly tested by analysis of E delta occupancy and function in versions of the minilocus and the endogenous locus. Novel cis- regulatory elements, such as recently discovered MARs that flank E delta, and a postulated regulatory element associated with D delta 3, will be tested for roles in the activation of TCR delta gene rearrangement as well. Finally, versions of minilocus and the endogenous locus will be analyzed to determine whether E delta and E alpha serve as developmental stage-and region-specific modulators of core histone acetylation, and whether the mechanisms of enhancer- blocking by BEAD-1 involves blockade of E delta-dependent core histone acetylation.